Chapter 3
Genetic Engineering

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Genes contain coded information that leads to the production of proteins.
Proteins, in turn, are responsible for creating the traits that
characterize individual organisms. Therefore, if a way could be found to
transfer genes from one organism to another, creatures could be
manufactured with traits that they had never before exhibited. Based on
the description of the structure of DNA provided by Watson and Crick,
researchers began to search for a way to cut genes from the DNA of one
organism and paste them into another. By the 1970s, they had the answer,
and the science of genetic engineering was born. It was a giant step
forward. Now, a mere thirty years later, it is possible to exchange genes
between one plant and another and one animal and another. It is even
possible to transpose genes between plants and animals. No
organism—from primitive life-forms, like bacteria, to higher order
animals, like human beings—is exempt from this genetic swap meet.
Genetic engineering has led to monumental advances in medicine and
agriculture, but it has also given rise to a storm of controversy and
debate over the limits on humankind's intrusion into the natural
order of things.

Restriction Enzymes and Plasmids

The first major breakthrough on the road to genetic engineering came with
work done on restriction endonucleases
by Herbert Boyer of the University of California at San Francisco. As
defined by Karl Drlica in
Understanding DNA and Gene Cloning: A Guide for the Curious
, restriction endonucleases "are a group of enzymes [a special type
of protein] that . . . occur naturally in a large number of different
bacterial species, serving as part of the natural defense mechanism that
protects bacterial cells against invasion by foreign DNA molecules such as
those contained in viruses."
15

When, for example, a virus attacks a single-celled bacterium, restriction
endonucleases are unleashed and go to work, cutting the invading DNA into
small, nonthreatening pieces. "Crucial to this protective device is
the ability of the nuclease to discriminate between its own DNA and the
invading DNA; otherwise the cell would destroy its own DNA," Drlica
says.

This recognition process involves two elements. First there are specific
nucleotide sequences [As and Ts, Cs and Gs] that act as targets for the
nuclease. These are called the restriction sites. Second, there is a
protective chemical signal that can be placed by the cell on all the
target sequences that happen to occur in its own DNA. The signal
modifies the DNA and prevents the nuclease from cutting. Invading DNAs,
lacking the protective signal, would be chopped by the nuclease.
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Thus, restriction enzymes have the remarkable ability to recognize
specific arrangements of DNA base pairs—As and Ts, Gs and Cs. They
also have the capacity to act like a molecular scalpel, severing the DNA
at exactly the spot where they detect this sequence of genetic letters.
Restriction enzymes are a powerful tool because there are thousands of
them, and each one acts only on a unique arrangement of As and Ts and Cs
and Gs.

A second piece of the genetic engineering puzzle fell into place when it
was discovered that bacteria

have another interesting property. Under the right conditions, small,
circular pieces of DNA can be transferred from one bacterial cell to
another. These DNA structures, called plasmids, are not located on the
bacterium's solitary chromosome, but float freely in other parts of
the organism. Single-cell bacteria duplicate when the cell divides,
producing an exact copy of itself. During this process, its plasmids, as
well as its chromosomal DNA, are also reproduced. "In 1959,
Japanese doctors found that the ineffectiveness of antibiotics as a cure
for dysentery with some patients was due to the fact that the bacteria
with which the patients were infected carried a plasmid containing several
genes of resistance to different antibiotics," says geneticist
Maxim D. Frank-Kamenetskii. "It was discovered that genes
of resistance to antibiotics are always carried by plasmids. An ability
to move freely between bacteria enables the plasmids carrying such genes
to spread rapidly among bacteria immediately in the wake of a broad
application of the antibiotic."
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Recombinant DNA Technology

Boyer and Stanley Cohen, another scientist at the University of California
who was working on plasmids, pooled their knowledge to conduct a series of
experiments on two different strains of the E. coli bacteria. Some forms
of E. coli live in the intestines of humans and other animals, where they
aid the body's digestive processes. Boyer and Cohen marshaled
restriction endonucleases to cut some E. coli plasmids. When plasmids are
cut, they leave what researchers call "sticky ends," to
which other plasmid segments can easily attach themselves. The point at
which the pieces of the two plasmids join is cemented by the activity of
an enzyme called ligase, which can be described as molecular glue, to form
a stable chemical bond. Then, the two scientists severed particular genes
from another type of bacteria, one that was resistant to antibiotics, and
spliced them to the sticky ends of the cut E. coli plasmids. The result: a
hybrid form of antibiotic-resistant
E. coli.

One big question remained to be answered. Thus far,

Dr. Stanley Cohen was one of two scientists who first experimented
with an antibiotic-resistant E. coli hybrid.

genes had been successfully exchanged between two types of bacteria, but
would the same cut-and-paste technique work when the genes came from two
radically different life-forms? In other words, could genes cross species
boundaries? Cohen's and Boyer's first attempt to transplant
genes from one form of life into another involved a tadpole and E. coli
bacteria. The scientists removed a gene from one of the tadpole's
cells and transplanted it into an
E. coli bacterial cell. When the bacterial cell started to multiply, the
scientists analyzed each successive generation and found that they all
contained the tadpole gene. The first gene transfer between species had
been accomplished, and the door was now open to a wide range of similar
experiments—many of them far more controversial. It had been
practically demonstrated that genes from fish, even genes from plants,
could be transplanted into humans.

The new technique was called recombinant DNA technology—just
another name for genetic engineering—because the procedure
recombined genes that originated in different organisms. The popular media
gave it another name that has been responsible for a great deal of
confusion. They called it gene cloning, creating the belief that science
could duplicate entire organisms, an achievement that was not at that
point even distantly attainable. But what the technique did allow
scientists to do was create specific types of proteins in large
quantities. "Usually, a specific protein is produced by a cell in
very small quantities, sometimes a mere one or two molecules per
cell," says Frank-Kamenetskii.

As a result, the production of proteins needed for particular research
was an arduous and costly undertaking. One had to process dozens of
kilograms [a unit of weight equal to 2.2 pounds], nay tons, of biomass
to obtain milligrams of protein.

Despite such meager quantities, it was still not possible to ensure the
necessary purity of the protein. Hence, the cost of many protein
preparations was exorbitant and their purity was substandard. Genetic
engineering brought about a radical change in this situation.
Genetic-engineering strains now exist—superproducers of many
proteins with high standards of purity—that were undreamed of
before. Molecular biology firms have sharply diversified the production
of enzymes and other protein preparations and have reduced the prices of
these products. Thus, molecular biology received a powerful new impetus,
resulting in an unheard of acceleration in the pace of scientific
research.
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In accomplishing this goal, bacteria, especially E. coli bacteria, have
proved to be the most effective host for transplanted genes because they
reproduce rapidly. For example, if scientists wish to produce a certain
kind of protein, they snip the required genes from an animal that produces
the protein naturally and transplant them into an E. coli cell. They then
put the cell in an environment that encourages it to divide and just let
nature take its course until they have millions of cells all producing the
desired protein. Finally, the scientists extract the protein from the
cells and use it for whatever purpose they have in mind.

Biofoods

The first area in which the new science of genetic engineering took hold
was agriculture. It quickly became apparent that food plants could be
genetically altered so they were more resistant to pests, needed less
water to grow, and provided more nutrition than in their natural states.
Since human beings first began to till the land, farmers have been trying
to produce hardier, more profitable crops. The method

Thanks to advances in genetic engineering, farmers can now
crossbreed crops like these corn plants to produce foods that are
healthier and hardier than those that grow naturally.

at their disposal was called selective breeding. In the same way that
Gregor Mendel bred pea plants to yield other pea plants with certain
traits, agriculturists crossbred the healthiest plants with each other to
create the most productive varieties possible. But the process took a long
time and, because the relationship between genes and traits is complex,
often led to unwanted results.

Genetic engineering takes the guesswork out of this effort and greatly
reduces the time it takes to produce a plant with the desired traits. It
also—for the first time—makes it possible to breed entirely
different types or species of plants with each other to create some truly
novel hybrids. Previously, selective breeding limited farmers to
experiments with plants of the same or very closely related species. By
cutting and pasting genes from one plant to another, genetic engineers are
able to do all the things that crossbreeding can do, and do them faster
and more accurately.

The technique also allows scientists to do things that nature alone is
incapable of doing. A report prepared at University of Virginia on the
state of currently available genetically engineered, or transgenic, plants
states:

Many plants have been commercialized, including tomatoes and squash and
commodity crops like corn and soybeans. Most have been engineered for
one of three traits: herbicide [weed killer] tolerance, insect
resistance, or virus tolerance. This is the fastest growing area of
biotechnology in agriculture. Genetically engineered cotton has been
approved for commercial use. There are between 10 and 12 million acres
of cotton in the U.S. and estimates are that all of this acreage will be
planted to transgenic varieties within the next 10 years. One of the
newest innovations in cotton is the development of naturally-colored
cotton fibers where the pigments have come from inserting color genes
from flowers into cotton.
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As a further indication of the widespread use of genetic engineering in
farming, the Food and Agriculture Organization of the United Nations,
citing figures for the year 1999 (the process of gathering such
information accurately is slow when developing countries are involved),
reports:

Transgenic plants . . . now cover large areas in certain parts of the
world. Estimates for 1999 indicate that 39.9 million hectares [a unit of
measurement equivalent to 2.47 acres] were planted with transgenic
crops. . . . Of the 39.9 million hectares, 28.1 million (i.e. 71%) were
modified for tolerance to a specific herbicide (which could be sprayed
on the field, killing weeds while leaving the crop undamaged); 8.9
million hectares (22%) were modified to include a toxin-producing gene
from a soil bacterium . . . which poisons insects feeding on the plants,
while 2.9 million hectares (7%) were planted with crops having both
herbicide tolerance and insect resistance.
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The bioengineering of plants has become big business. Hundreds of millions
of dollars of research money are being poured into a diverse range of
projects. Among the most promising are creating plants that produce their
own fertilizer and modifying plants to be delivery systems for medicines
and nutrients they do not naturally produce. For example, work is under
way to produce a banana that contains in its DNA a vaccine for hepatitis
B, a highly contagious disease that damages the livers of people who
contract it. The banana is also being turned into a megavitamin to deliver
much-needed nutrients to children in the underdeveloped countries of
Africa and Asia.

Transgenic Animals

Plants are not the only organisms that genetic engineers are working on.
Turning their attention to animals, scientists have produced a number of
transgenic creatures they hope will bring major benefits to mankind. For
example, human genes have been put into pigs to allow the pigs to produce
human insulin, a substance needed to control diabetes, one of the
fastest-growing diseases in affluent countries.

The applications are wide-ranging. Goats are being genetically modified to
produce a protein that aids in blood clotting. Other experiments with
goats aim to find cures for multiple sclerosis and some forms of cancer.
Sheep are being altered to generate a protein that may fight the lung
disease emphysema. Designer dairy products are also on the drawing boards.
Geneticists hope to end up with a breed of cow that produces, for example,
only low-fat milk.

Other genetically engineered animals are being designed to contract human
diseases so that experimental treatments can be explored. In these cases,
healthy genes are replaced with malfunctioning counterparts, using a
technique similar to the cut-and-paste procedure used with plasmids.

Finally, researchers are optimistic that they will be able to turn
animals, principally pigs, into sources of organs for human transplants.
To accomplish this, they are transferring human genes into pigs so that
the resulting organs will more closely resemble those found in humans and
thus be less likely to be rejected.

Cloning

Work on transgenic animals has also led to the cloning of entire
organisms. A clone is an identical genetic copy of an organism—its
DNA is the same as that of the original from which the copy was made. In
humans, identical twins are naturally occurring clones. In these cases,
the original fertilized egg divides into two genetically identical halves
and proceeds to develop into two distinct babies. Since the babies
originated from the same egg fertilized by the same sperm, they have
exactly the same DNA. By contrast, fraternal twins come from two separate
eggs, each of which is fertilized by a different sperm cell. Even though
these children are born at the same time, they are as genetically
different from each other as any other pair of siblings born years apart
would be.

The cloning of organisms must be carefully distinguished from the cloning
of genes—a distinction that the popular media have not always
succeeded in making. The cloning of single genes, using plasmids, is an
established procedure; the cloning of organisms is still experimental and
highly controversial. Although several fringe groups claim to have
successfully cloned human beings, they have failed—as of the
writing of this book—to produce any evidence to support their
contentions.

To this date, the most famous cloned mammal remains Dolly the sheep. A
close look at how Dolly was created will provide a good description of the
techniques required to clone any higher animal, including humans. In
announcing Dolly's birth in 1997,
Scientific American
magazine reported:

Dolly, unlike any other mammal that has ever lived, is an identical copy
of another adult and has no father. She is a clone, the creation of a
group of veterinary researchers. That work, performed by Ian Wilmut and
his colleagues at the Roslin Institute in Edinburgh, Scotland, has
provided an important new research tool and has shattered a belief
widespread among biologists that cells from adult mammals cannot be
persuaded to regenerate a whole animal.
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Previously, researchers had cloned mammals and other animals using
embryonic cells as a starting point. Embryonic cells, taken from an
undeveloped fertilized egg, are different from adult cells in that they
are undifferentiated. When an egg cell is fertilized, it starts to divide.
Up to a certain point, the cells in each succeeding generation have the
ability to develop into specialized cells that will make up the various
parts (organs, bones, skin, etc.) of the mature organism. After that
point, cells become differentiated, or specialized—some of them
begin to turn into liver cells, others into brain cells, and so
on. Until Dolly, it was thought that clones could be produced only from
undifferentiated cells that would divide and grow to maturity as the
cloned organism developed.

Promise and Problems of Cloning

Dolly, however, was cloned from a cell taken from the udder of a
six-year-old female sheep, a fully developed adult. Dolly was the 277th
attempt made by Wilmut and his fellow researchers. The other attempts had

Scotland's Dolly the sheep was the world's first
cloned mammal.

failed, but the same technique was used in all of them. "Wilmut
and his co-workers accomplished their feat by transferring the nuclei from
various types of sheep cells into unfertilized sheep eggs from which the
natural nuclei had been removed by microsurgery," the
Scientific American
article continues.

Once the transfer was complete, the recipient eggs contained a complete
set of genes, just as they would do if they had been fertilized by
sperm. The eggs were then cultured for a period before being implanted
into sheep that carried them to term, one of which culminated in a
successful birth. The resulting lamb was, as expected, an exact genetic
copy, or clone, of the sheep that provided the transferred nucleus, not
of [the sheep] that provided the egg.
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Wilmut used a pipette many times thinner than a human hair to remove the
DNA from the host egg. Then the empty egg was placed next to a cell taken
from the donor sheep's udder and the two were fused together using
a tiny jolt of electricity. Another pulse of electricity caused the egg
cell, with its new DNA, to start dividing. The cell was now behaving just
like a normal egg cell would if it had been fertilized by sperm from a
male sheep. It was cultured for a few days in a laboratory dish and then
implanted into the uterus of a third sheep, which carried it to term and
gave birth to Dolly.

From the beginning, Wilmut and other geneticists were concerned that since
Dolly's DNA came from a six-year-old sheep she might age
prematurely. At first, she seemed to be perfectly healthy and gave birth
to a lamb of her own in 1998. But then Dolly began to develop medical
problems frequently associated with aging. "Early in life Dolly had
a weight problem. Then in 1999, it emerged that caps at her chromosome
ends called telomeres, which get shorter each time a cell divides, were 20
percent shorter than

was normal for a sheep her age," says science journalist John
Whitfield, writing in the journal
Nature.
"This led to speculation that Dolly's biological age might
equal that of her and her mother combined."
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In 2002, Dolly was diagnosed with arthritis, another disease associated
with old age, and then she came down with lung cancer. Dolly was humanely
put to death at the age of six and a half years, half the normal life span
of a sheep of her kind. Cloned animals since Dolly, including cows,
rabbits, mice,
cats, goats, and pigs, have experienced similar problems.
"Dolly's premature death is typical of cloned
animals," Whitfield writes.

From conception onwards, clones suffer a higher mortality rate than
non-clones. Studies in mice seem to show that this bad health persists
throughout life. Some seized upon Dolly's ailments as evidence
that clones are invariably sickly and age prematurely. Although it
can't be ruled out that her origins made her less robust than
other sheep, it is not possible to make generalizations about
clones' health from the fate of a single animal. . . . But the
process of genetic reprogramming seems too complex and haphazard to
control tightly, and its success rate has not improved much since
Dolly's day.
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Although many questions about animal cloning remain to be answered,
scientists are hopeful that, as

Genetically engineering oversized cattle to increase beef yield is
just one of the potential benefits of cloning.

with genetically engineered plants, there are many benefits in store.
Among these are creating oversized cattle to improve beef yield and the
production of stem cells, multipurpose cells found in embryos, which may
prove to have a wide range of medical applications. Like transgenic
plants, transgenic animals can be created to produce greater amounts of
nutrients, and some scientists also claim that cloning can be used to
preserve endangered species from extinction.

The Human Genome Project

The human genome is composed of about 3 billion base pairs of As and Ts,
Gs and Cs. In 1990, an international consortium of scientists set out to
create a map that would show exactly where on our twenty-three pairs of
chromosomes every one of those base pairs is located. The effort, called
the Human Genome Project, is the most extensive scientific enterprise ever
undertaken.

Genetic mapping is the first step in isolating a gene. There are two basic
approaches to this complex endeavor. The first is called linkage mapping,
and its goal is to show where on each chromosome each gene is located
relative to other genes. The method is to compare the genetic makeup of
members of a family or closely related group of people who have a history
of a specific disease. By finding similar base pairs on the chromosomes of
family members in succeeding generations of this group who have the
disease, scientists can isolate the responsible gene and begin to build a
map of the entire genome. The second type of map is far more ambitious. It
is called a physical map and its goal is to show the absolute (not
relative) location of the base pairs that make up every gene.

In both cases, the cut-and-paste techniques developed by genetic
engineering are indispensable. Restriction enzymes are used to cut the
chromosomes
into small segments, which are then cut into even smaller pieces until
the sought-after gene is found. It is a painstaking process, made possible
not only by the methods of genetic engineering, but also by powerful
supercomputers that enable researchers to compare various overlapping
segments to weed out duplicate base pairs and base pairs that appear not
to play any role in the process of genetic inheritance. Only about 2
percent of the 3 billion base pairs actually make up functional genes. The
rest, called junk DNA, help to locate the genes and may play other roles
that remain to be determined.

The Human Genome Project was virtually completed in the year 2003. It
yielded many interesting—and surprising—insights into the
genetic composition of human beings. Among them: the total number of genes
in a human being lies between 30,000 and 35,000, far fewer than earlier
estimates of 80,000 to 140,000; the average gene consists of about 3,000
base pairs, but sizes vary greatly, the longest being 2.4 million base
pairs; 99.9 percent of base pairs are identical in all people; the
function of more than 50 percent of human genes has not yet been
determined. While work continues on tracking down the roles played by
these mystery genes, the next step is the mapping of the human
proteome—the complete array of proteins operating in the human
body. This includes pinpointing all the proteins coded for by the genes
and describing the specific role these proteins play in the human
organism.

really helpful website. used it for my recombinant DNA science essay. great help as it has almost all the info i needed. might be worth adding in a bit about advantages and disadvantages of genetic engineering and also a bit about the ethical issues.

Has recombinant dna succeeded in creating new viable species? Has the technique proved Evolution as a fact? If it has created new species, are these species fertile and will they produce eventually, unaided, new genera, phylum etc